Tocotrienols: Exciting Biological and Pharmacological Properties of Tocotrienols and other Naturally Occurring Compounds, Part I

Inflammation has been implicated in cardiovascular disease and tocotrienols are potent hypocholesterolemic agents that reduce β-hydroxy-β-methyl-glutaryl coenzyme A reductase activity, which is degraded via the ubiquitin-proteasome pathway. Impact of various tocotrienols (α-, γ-, or δ-tocotrienol) treatments inhibit the chymotrypsin-like activity of 20S rabbit muscle proteasome (>50%) in RAW 264.7 cells and BALB/c mice. Moreover, the effect of various tocotrienols (α-, γ-, or δ-tocotrienol), α-tocopherol, quercetin, riboflavin, (−) Corey lactone, amiloride, dexamethasone supplemented diets fed to chickens (4-weeks) resulted in reduction of total cholesterol, LDL-cholesterol, and triglycerides. This trend was also observed in macrophages from RAW 264.7 cells, in LPS-induced thioglycolate-elicited peritoneal macrophages derived from C57BL/6, BALB/c, LMP7/MECL-1−/−, and PPAR-α−/− knockout mice from young (4-week-old) and senescent (42-week-old) mice, resulting in significant inhibition of TNF-α and nitric oxide levels (30% to 70%), blocked degradation of P-IκB protein, and decreased activation of NF-κB, followed gene suppression of mRNA levels of TNF-α, IL-1β, IL-6, and iNOS. In human study, normal or hypercholesterolemic subjects administered two capsules/d of NS-7 or NS-6 (4-weeks) showed decrease in serum CRP, NO, γ-GT, total cholesterol, LDL-cholesterol, and triglycerides levels in normal as compared to hypercholesterolemic subjects (12% to 39%). In second study, hypercholesterolemic subjects were given increasing doses of δ-tocotrienol (125 mg, 250 mg, 500 mg, and 750 mg/day) plus AHA Step-1 diet (4-weeks). The most effective dose of tocotrienols (250 mg/day) may be used to lower serum NO (40%), CRP (40%), MDA (34%), γ-GT (22 %), and inflammatory cytokines IL-1α, IL-12, IFN-γ by 15% to 17%, and increase TAS levels by 22%.


Introduction
All human studies were double-blind, randomized, placebo-controlled trial (RCT). A nonprobability convenience sampling technique was used. The protocol of each human study was registered with WHO regional office in Asia (World Health Organization Sri Lanka Clinical Trial Registry, Sri Lanka Center; srilanactr@gmail.com, after ethical approval by the Institutional Review Board of Armed Forces Institute of Pathology (AFIP), Rawalpindi, Pakistan. The registry number and date has been reported in each human study paper. The studies were carried out according to the guidelines provided by the United States Food and Drug Administration (FDA, 2003) at (AFIP), Rawalpindi, and National University of Medical Sciences, Rawalpindi, Pakistan. All participants of human studies have signed an informed consent form before start of the study. All papers were published in refereed journals.

Anti-Inflammatory and Hypocholesterolemic Properties of Tocotrienols and Other Compounds in Various Experimental Models
As mentioned earlier, inflammation has been implicated in cardiovascular disease, and the important role of proteasome in the development of inflammation and other macrophage functions has been demonstrated by us [14][15][16]. Lipopolysaccharide (LPS) was also used as a prototype for inflammation [17]. Tocotrienols are potent hypocholesterolemic agents that inhibit β-hydroxy-β-methylglutaryl coenzyme A reductase activity, which is degraded via the ubiquitin-proteasome pathway. Our results have demonstrated the impact of various tocotrienols (α-, γ-, or δ-tocotrienol; Figure 1) treatments inhibit the chymotrypsin-like activity of 20S rabbit muscle proteasome (>50%) in murine RAW 264.7 cells and BALB/c mice ( Figure 2A). Furthermore, chymotrypsin-like, trypsin-like, post-glutamase activities were decreased >40% with low concentrations (<80 μM) and increased gradually (80 μM to 640 μM) in RAW 264.7 cells and BALB/c mice ( Figure 2B) [18]. Moreover, tocotrienols showed 9% to 33% inhibition in TNF-α secretion in LPS-stimulated RAW 264.7 cells ( Figure 2C). Serum levels of TNF-α with LPS-induced secretion were reduced (20% to 48%) by tocotrienols with the doses of 1 and 10 μg/kg in mice ( Figure 2D), and a corresponding rise was observed in serum levels of corticosterone (19% to 41%) and adrenocorticotropic hormone (81% to 145%) ( Figure 3A, 3B). The maximum inhibition was observed with δ-tocotrienol (10.0 μg/kg) [18]. The low concentrations of δ-tocotrienols (<20 μM) blocked LPS-induced gene expression of TNF-α, IL-1β, IL-6 and iNOS (>40%), and increases with higher concentrations (40 μM) in peritoneal macrophages prepared from BALB/c mice compared to control group ( Figures 3C, 3D) [18]. These results represent a novel approach of proteasome modulators, which may lead to the development of new dietary supplements for cardiovascular and other human diseases based on inflammation [18].
Our results on evaluation of δ-tocotrienol and quercetin on inflammatory markers and lipid parameters was reported in Chickens. It is well known that inflammatory responses to a wide variety of stimuli are largely attributable to up-regulation of the pro-inflammatory transcription nuclear factor kappaB (NF-κB). Specifically, Reactive Oxygen Species (ROS) up-regulate the pro-inflammatory NF-κB transcription factor [19]. The increased transport of NF-κB to the cell nucleus enhances expression of numerous genes encoding proteins that contribute to the inflammatory process, including inducible Nitric Oxide Synthase (iNOS), Cyclooxygenase-2 (COX-2), Tumor Necrosis Factors (TNF-α, TNF-β), Interleukins (IL-1, IL-6), chemokines (IL-8, MCP1, and MIP1α), Activator Protein-1 (AP-1) and adhesion factors (ICAM, and VCAM). Several of the proteins encoded by genes that are up-regulated by NF-κB are also potent NF-κB activators [19]. A series of in vitro tests confirmed the strong anti-inflammatory activities of known inhibitors of NF-κB activation (δ-tocotrienol, quercetin, riboflavin, (−) Corey lactone, amiloride, and dexamethasone; Figure 4 [20]). As it was demonstrated that δ-Tocotrienol suppresses β-Hydroxy-β-Methylglutaryl Coenzyme A (HMG-CoA) reductase activity, and concomitantly lowers serum total and LDL cholesterol levels [8]. The results of above compounds ( Figure 4) were reported in an avian model anticipating that a dietary additive combining δ-tocotrienol with quercetin, riboflavin, (−) Corey lactone, amiloride, or dexamethasone would yield greater reductions in serum levels of total cholesterol, LDL-cholesterol, and inflammatory markers (Tumor Necrosis Factor-α [TNF-α], and Nitric Oxide [NO]), than that attained with the individual compounds [21].
In these series of experiments, the control diet was supplemented individually with compounds mentioned above ( Figure 4) and fed to chickens for 4-weeks, the riboflavin, (−) Corey lactone, δ-tocotrienol, and quercetin produced small reductions in body weight gains as compared to control. Whereas dexamethasone significantly and markedly reduced weight gain (>75%) compared to control [21]. The serum levels of TNF-α and NO were decreased 61% to 84% and 14% to 67%, respectively in chickens fed diets supplemented with δ-tocotrienol, quercetin, riboflavin, (−) Corey lactone, amiloride, or dexamethasone as compared to controls ( Figure 5A, 5B). Furthermore, significant decrease in the levels of serum total and LDL-cholesterol was observed with δ-tocotrienol, quercetin, riboflavin and (−) Corey lactone (13% to 57%; Figure 5C, 5D), and these levels were 2-fold higher in dexamethasone treated chickens as compared to controls [21]. Moreover, the combination of δ-tocotrienol with the other compounds yielded even lower levels of these markers as compared to individual components [21]. Exceptions were significantly lower total and LDL cholesterol and triglycerides values attained with the δ-tocotrienol + (−) Corey lactone treatment and the significantly lower triglycerides value attained with the δ-tocotrienol + riboflavin treatment. Thus δ-tocotrienol attenuated the lipid-elevating impact TNF-α levels decreased by >60% by each of the experimental compounds. Additionally, all the treatments except for dexamethasone resulted in lower serum total cholesterol, LDLcholesterol, and triglycerides levels. The impact of above-mentioned compounds on the factors evaluated herein was increased when combined with δ-tocotrienol, thus δ-tocotrienol is the most potent inhibitor for inflammation [21].
The results also indicated that intravenously administered tocotrienols were significantly better than tocopherols in inhibiting Cyclic Flow Reduction (CFRs), a measure of acute platelet-mediated thrombus formation. Tocotrienols given intravenously (10 mg/kg), abolished CFRs after a mean of 68 min (range 22 min to 130 min), and this abolition of CFRs was sustained throughout the monitoring period (50 min to 160 min) [21]. The pharmacokinetic results indicated that treatment with α-tocopherol increased levels of total tocopherols in post-treatment vs. pre-treatment specimens (57 μg/mL vs. 18 μg/mL in plasma and 42 μg/mL vs. 10 μg/mL in platelets (Table 2A). However, treatment with α-tocopherol resulted in slightly decreased levels of tocotrienols in post-treatment vs. pretreatment samples (1.4 μg/mL vs. 2.9 μg/mL in plasma and 2.3 μg/mL vs. 2.8 μg/mL in platelets (Table 2A) [22].
However, treatment with α-tocotrienol increased levels of both tocopherols and tocotrienols in post-treatment vs. pre-treatment samples (tocopherols, 45 μg/mL vs. 10 μg/mL in plasma and 28 μg/mL vs. 5 μg/mL in platelets; tocotrienols, 2.8 μg/mL vs. 0.9 μg/mL in plasma and 1.28 μg/mL vs. 1.02 μg/mL in platelets (Table 2B). The TRF treatment also increased levels of tocopherols and tocotrienols in post-treatment vs. pre-treatment samples (tocopherols, 68 μg/mL vs. 20 μg/mL in plasma and 31.4 μg/mL vs. 7.9 μg/mL in platelets, tocotrienols, 8.6 μg/mL vs. 1.7 μg/mL in plasma and 5.8 μg/mL vs. 3.9 μg/mL in platelets, (Table 2C) [22]. These results indicated that intravenously administered tocotrienols inhibited acute platelet-mediated thrombus formation, and collagen and ADPinduced platelet aggregation. Tocotrienol treatment induced increase in α-tocopherol levels of 2-fold and 4-fold in plasma and platelets, respectively. Interestingly, tocotrienol treatment induced a less pronounced increase in the levels of tocotrienols in plasma and platelets, suggesting that intravenously administered tocotrienols may be converted to tocopherols. Tocotrienols, given intravenously, could potentially prevent pathological platelet thrombus formation and provide a therapeutic benefit in conditions such as stroke and myocardial infarction [22].
It is well known that as humans age, they are at increased risk for a variety of ageassociated diseases such as cardiovascular, arthritis, diabetes, obesity, dementia, cancer, and atherosclerosis [23]. Over the past decade, it has become increasing evident that dysregulated immune function, leading to chronic inflammation, which contributes to the pathogenesis of several of these age-associated disease [24].
These five compounds also suppressed LPS-induced secretion of TNF-α in macrophages obtained from C57BL/6 and BALB/c mice [25]. The TNF-α secretion, however, was not suppressed by any of these three proteasome inhibitors tested (δ-tocotrienol, riboflavin, and quercetin) with LPS-stimulated macrophages from LMP7/MECL-1 −/− and PPAR-α −/− knockout mice. Results of gene expression for TNF-α and iNOS were generally consistent with results obtained for TNF-α protein and NO production as observed with four strains of mice ( Figures 8A-8D) [25].
These results have indicated that δ-tocotrienol, riboflavin, and quercetin inhibit NO production in LPS-stimulated macrophages of all four strains of mice, and TNF-α secretion only in LPS-stimulated macrophages of C57BL/6 and BALB/c mice [25]. The mechanism for this inhibition appears to be decreased proteolytic degradation of P-IκB protein due to the inhibition of proteasome, resulting in decreased translocation of activated NF-κB to the nucleus, and depressed transcription of gene expression of TNF-α, and iNOS. Furthermore, these naturally occurring proteasome inhibitors appear to be relatively potent inhibitors of multiple proteasome subunits in inflammatory proteasomes. Consequently, these agents could potentially suppress the production of inflammatory mediators in ageing humans, thereby decreasing the risk of developing a variety of age-related diseases [25].
In recent years, the concept that age-associated diseases such as cardiovascular, cancer, dementia might be attributable, in part, to dysregulated inflammatory responses has been the subjects of extensive discussion [26]. Changes in immune function believed to contribute to a variety of age-related diseases, which have been associated with increased production of Nitric Oxide (NO). It was recently reported that dexamethasone, mevinolin, quercetin, δ-tocotrienol, and riboflavin ( Figure 4); can inhibit Lipopolysaccharide (LPS)-induced NO production in vitro in RAW 264.7 cells and in thioglycolate-elicited peritoneal macrophages derived from four strains of mice (C57BL/6, BALB/c, LMP7/MECL-1 −/− and PPAR-α −/− knockout mice) [25]. The potential results of the effects of diet supplementation with naturally occurring compounds (δ-tocotrienol and quercetin) on LPS-stimulated production of NO, TNF-α, and other pro-inflammatory cytokines involved in the ageing process have been reported. Young (4-week-old) and senescent mice (42-week-old) were fed control diet with or without quercetin (100 ppm), δ-tocotrienol (100 ppm), or dexamethasone (10 ppm; included as positive control for suppression of inflammation) for 4-weeks ( Figure 4). The thioglycolate-elicited peritoneal macrophages were collected, stimulated with LPS, LPS plus Interferon-β (IFN-β), or LPS plus Interferon-γ (IFN-γ), and inflammatory responses assessed as measured by production of NO and TNF-α, mRNA down-regulation for TNF-α, and iNOS genes, and microarray analysis [27].
The results of thioglycolate-elicited peritoneal macrophages prepared after four weeks of feeding of these mice, and then challenged with LPS (10 ng or 100 ng) resulted in increases of 55% and 73%, respectively in the production of NO of 46-week-old compared to 8-weekold mice fed control diet alone (respective control groups), without affecting the secretion of TNF-α among these two groups [27]. However, macrophages obtained after feeding with quercetin, δ-tocotrienol, and dexamethasone significantly inhibited (30% to 60%) the LPS-stimulated NO production, compared to respective control groups ( Figures 9A-9D). There was a 2-fold increase in the production of NO, when LPS-stimulated macrophages of quercetin, δ-tocotrienol, or dexamethasone were also treated with IFN-β or IFN-γ compared to respective control groups. It has been also demonstrated that NO levels and iNOS mRNA expression levels were significantly higher in LPS-stimulated macrophages from senescent (0.69 vs. 0.41), compared to young mice. In contrast, age did not appear to impact levels of TNF-α protein or mRNA expression levels (0.38 vs. 0.35) in LPS-stimulated macrophages (Table 3) [27]. These results have demonstrated that quercetin and δ-tocotrienols inhibit the LPS-induced NO production in vivo. The microarray RNAs analyses, followed by pathway analyses indicated that quercetin or δ-tocotrienol inhibit several LPS-induced gene expressions of several ageing and pro-inflammatory genes (IL-1β, IL-1α, IL-6, TNF-α, IL-12, iNOS, VCAM1, ICAM1, COX2, IL-1RA, TRAF1 and CD40) as shown in Table 3 [27]. The NF-κB pathway regulates the production of NO and inhibits the pro-inflammatory cytokines involved in normal and ageing process. The ex vivo results confirmed the earlier in vitro findings. These results of inhibition of NO production by quercetin and δ-tocotrienol may be of clinical significance treating several inflammatory diseases, including ageing process [27].
It was reported earlier that altered immune function during ageing results in increased production of Nitric Oxide (NO) and other inflammatory mediators. Recently, it was also reported that NO production was inhibited by naturally occurring quercetin, δ-tocotrienol, and riboflavin in Lipopolysaccharide (LPS)-stimulated RAW 264.7 cells, and thioglycolateelicited peritoneal macrophages from C57BL/6 mice ( Figure 4). In our continuous effort to find more potent, non-toxic, commercially available, naturally occurring compounds that suppress inflammation, the results of study have reported the inhibition of NF-κB activation and NO, TNF-α, IL-6, IL-1β, and iNOS gene expression by trans-resveratrol, transpterostilbene, morin hydrate, and nicotinic acid in LPS-induced RAW 264.7 cells and thioglycolate-elicited peritoneal macrophages from C57BL/6 and BALB/c mice [28].
These results have demonstrated that resveratrol and pterostilbene are particularly potent compounds that suppress expression of genes, and production of inflammatory products in LPS-stimulated RAW 264.7 cells, and macrophages from C57BL/6 and BALB/c mice [27]. Resveratrol and pterostilbene which are present in grapes, blueberries, and red wine, have been implicated as contributing factors to the lower incidence of cardiovascular disease in the French population, despite their relatively high dietary fat intake. Consequently, it appears likely that the beneficial nutritional effects of resveratrol and pterostilbene are due at least in part, to their ability to inhibit NF-B activation by the proteasome inhibitors, thereby suppressing activation of pro-inflammatory cytokines and iNOS genes, resulting in decreased production of TNF-α, IL-1β, IL-6, and NO levels, in response to inflammatory stimuli. This was the first report that has demonstrated that resveratrol and pterostilbene act as proteasome inhibitors, thus providing a mechanism for their anti-inflammatory effects in mouse cells [28].

Hypocholesterolmic and Anti-Inflammatory Properties of Tocotrienols and Other Compounds in Humans
It was earlier reported, the anti-inflammatory properties of resveratrol, pterostilbene, morin hydrate, quercetin, δ-tocotrienol, riboflavin in a variety of experimental animal models, and these compounds act by inhibiting proteasomal activity [25]. As reported earlier that serum Nitric Oxide (NO) levels increase with age in humans, and we hypothesized that combined cholesterol-lowering and inflammation-reducing properties of resveratrol, pterostilbene, morin hydrate, quercetin, δ-tocotrienol, riboflavin, and nicotinic acid ( Figure  4) would reduce cardiovascular risk factors in humans when used as nutritional supplements with, or without, other dietary changes.
The elderly human subjects were divided into two groups based on total serum cholesterol levels. Initial total serum cholesterol levels were normal in group #1 and elevated in group #2 subjects, respectively, and after establishing the Baseline serum NO, C-Reactive Protein (CRP), γ-Glutamyltransferase (γ-GT) activity, uric acid, Total Antioxidant Status (TAS), total cholesterol, HDL-cholesterol, LDL-cholesterol, and triglycerides levels [29]. Group 1 subjects were administered nutritional supplementation with one of two different combinations (NS-7=25 mg of each, resveratrol, pterostilbene, quercetin, δ-tocotrienol, nicotinic acid, morin hydrate or NS-6=morin hydrate replaced with quercetin, 50 mg/ capsule). Group 2 subjects also received these nutritional supplements (two capsules/d), including an AHA Step-1 diet for group 2 subjects for 4-weeks [29].
Clinical studies using Tocotrienol-Rich Fraction (TRF) from palm oil yielded inconsistent results with regards to its efficacy due to presence of tocopherols in TRF mixture. The impact of tocopherol-free δ-tocotrienol ( There was significant down-regulation in pro-inflammatory cytokines and gene expressions of resistin, IL-2α, IL-6, IL-12, IL-18, TNF-α, and others, that are normally involved in pathogenesis of atherosclerosis, diabetes, and ageing processes (

Conclusions
The anti-inflammatory effects of δ-tocotrienol, quercetin, riboflavin, (−) Corey lactone, amiloride, and dexamethasone on serum TNF-α and NO levels has been reported for the first time. Additionally, all the treatments except with dexamethasone resulted in lower serum total cholesterol, LDL-cholesterol, and triglycerides levels. The mechanism for this inhibition appears to be decreased proteolytic degradation of P-IB protein due to the inhibition of proteasome, resulting in decreased translocation of activated NF-κB to the nucleus, and depressed transcription of gene expression of TNF-α, and iNOS. The impact of above compounds was increased on these parameters when combined with δ-tocotrienol.
Moreover, these results also indicated that intravenously administered tocotrienols inhibited acute platelet-mediated thrombus formation, and collagen and ADP-induced platelet aggregation. The δ-tocotrienol, riboflavin, and quercetin inhibit NO production in LPSstimulated macrophages of all four strains of mice, and TNF-α secretion only in LPSstimulated macrophages of C57BL/6 and BALB/c mice. Furthermore, these naturally occurring compounds are potent inhibitors of multiple subunits in the proteasomes. Consequently, these agents could potentially suppress the production of inflammatory mediators in ageing humans, thereby decreasing the risk of developing a variety of ageing related diseases.
δ-Tocotrienol and quercetin inhibit the LPS-induced NO production in vivo. The microarray RNA analyses, followed by ingenuity pathway analyses indicated that quercetin or δtocotrienol inhibit LPS-induced gene expression of several genes involved ageing and proinflammation, such as IL-1β, IL-1α, IL-6, TNF-α, IL-12, iNOS, VCAM1, ICAM1, COX2, IL-1RA, TRAF1 and CD40. The NF-κB pathway regulates the production of NO and the pro-inflammatory cytokines involved in normal and ageing process. The ex vivo results confirmed the earlier in vitro findings. Thus, these findings of inhibition of NO production by δ-tocotrienol and quercetin may be of clinical significance treating several inflammatory diseases, including ageing process. This was the first report demonstrating that resveratrol and pterostilbene act as proteasome inhibitors, thus providing a mechanism for their antiinflammatory effects. These results indicated that serum NO levels are elevated in elderly humans compared to children or young adults. Diet supplementation with combinations of resveratrol, pterostilbene, morin hydrate, quercetin, δ-tocotrienol, riboflavin, and nicotinic acid reduce cardiovascular risk factors in humans when used as nutritional supplements with, or without, other dietary changes.
In a dose-dependent study of 125 mg/day to 750 mg/day, δ-tocotrienol maximally reduced inflammation and oxidative stress parameters with a 250 mg/day dose in hypercholesterolemic subjects and may be an attractive therapeutic alternative for the natural maintenance of health during aging process. The plasma inflammatory miRNAs (miR-101a, miR-125a, miR-155, miR-223) were down-regulated as compared to pre-dose values. The miRNA-146a increased during senescence and treatment with these compounds down-regulated elevated levels of miRNA-146a.

2
Data are means ± SD, n = 3 mice per group.

3
Percentage of gain in weight in each group compared to respective control group are in parenthesis.

a-b
Values in a row not sharing a common superscript letter are significantly different at P < 0.05.
Ann Clin Case Rep. Author manuscript; available in PMC 2022 December 27.

Qureshi
Page 34 Effects of various compounds on the gene expression of TNF-α and iNOS in LPS-stimulated thioglycolate-slicited peritoneal macrophages derived from 8-week-old and 46-week-old C57BL/6 male mice after feeding for 4-weeks 1 .      The estimations of plasma nitric oxide (NO) were carried out according to published procedure as described in Materials and Methods.